Project Summary This application requests support for studies of coprophilous (dung-colonizing) fungi as sources of novel natural products (NPs) useful in developing new treatments for Cryptococcus fungal infections-- an area of unmet medical need. The project involves a collaboration between three highly qualified and productive research groups with a well matched array of complementary experience in discovery of new bioactive fungal NPs from both academic and industrial perspectives, in evaluation and development of antifungal agents, and in Cryptococcus pathogenesis. Reasons for targeting coprophilous fungi as sources of new antifungal NPs include ecological adaptations for microbial antagonism, genomic evidence for novel pathways, historical under-representation in screening programs, and positive results from earlier studies conducted independently by two of the Co-PIs. The approach seeks to avoid inefficiencies inherent in empirical microbial screening paradigms that rely on testing vast numbers of uncharacterized isolates. A unique, high-quality collection of taxonomically characterized fungi will be assembled. Strains will be isolated from dung collections from the Southwestern U.S.?a biodiverse region rich in coprophilous species. Strains will be cultured in arrays of media using methods selected on the basis of the Co-PI's extensive industrial fermentation experience. Extracts from the resulting cultures will be multimodally interrogated for anti-Cryptococcus activity using assays for growth inhibition, differential thermosensitivity, and synergy with existing antifungal drugs. Selected, NP-rich extracts will be subjected to pre-fractionation. Counterscreens will be used to de-prioritize extracts and fractions with significant mammalian cytotoxicity. A diverse subset of the most distinctive and productive species will be genome-sequenced, and gene inactivation strategies will be employed in efforts to remodel expression of their secondary metabolic products. Samples showing potent and specific antifungal effects will be subjected to assay-guided fractionation, and active agents will be identified using spectroscopic methods. Unique information about coprophilous fungal biology and phylogeny will emerge, and will be combined with assay data and chemical results to identify fungal lineages likely to afford further new antifungal NPs in ongoing studies. Active NPs of interest will be produced by scale-up protocols and subjected to in-depth evaluation, including testing in a validated invertebrate Cryptococcus disease model, and, where warranted, a mouse model. Mode-of-action studies will be undertaken for the most promising agents. This project will enable a thorough assessment of hypotheses regarding the promise of these distinctive fungi as sources of novel antifungal NPs, and offers considerable potential for discovery of innovative therapeutic agents effective against cryptococcal diseases.